CN103403159A - Method for producing virus vector for gene transfer - Google Patents
Method for producing virus vector for gene transfer Download PDFInfo
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- CN103403159A CN103403159A CN2012800080734A CN201280008073A CN103403159A CN 103403159 A CN103403159 A CN 103403159A CN 2012800080734 A CN2012800080734 A CN 2012800080734A CN 201280008073 A CN201280008073 A CN 201280008073A CN 103403159 A CN103403159 A CN 103403159A
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- C12N2760/18011—Paramyxoviridae
- C12N2760/18611—Respirovirus, e.g. Bovine, human parainfluenza 1,3
- C12N2760/18641—Use of virus, viral particle or viral elements as a vector
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Abstract
The following are disclosed in the present invention: a cell line capable of constantly and stably expressing F proteins, and acting as a host cell for the transmission of an F gene-defective virus, and a method for producing the F gene-defective virus using such cells. A replication-deficient human parainfluenza type 2 virus vector is produced by co-culturing an F gene-defective human parainfluenza type 2 virus with Vero cells having the F gene of the human parainfluenza type 2 virus in a form which is expressed non-inductively, and then isolating virus particles from the culture supernatant.
Description
Related application
The application advocates right of priority based on Japanese patent application 2011-025234 (application on February 8th, 2011), and the content of Japanese patent application 2011-025234 is by reference to incorporating the application into.
Technical field
The present invention relates to the manufacture method of the virus vector that imports for gene.
Background technology
Because human parainfluenza 2 C-type virus Cs (human parainfluenza type2virus) (hPIV2) likely infect people's tracheae mucous membrane, mucosa immunity-inducing, so expect that it applies with carrier as vaccine., for this carrier is practical as pharmaceuticals, must make virus vector have the ability of infection people cell and infect the rear propagated virus (being expressed as the non-proliferative carrier) that do not produce in human body.That is, cell tissue is carried out Single-infection, but do not produce propagated virus in the cells infected tissue, do not cause that repeatedly the system that infects is necessary., in order to build this system, usually adopt following system: make gene part defect on viral genome, make the cell of expressing this dcc gene product, by in this cell to the trans supply dcc gene of defective virus product, make non-proliferous type carrier.For DNA virus, imported this system (for example WO94/28152) in adenovirus,, for RNA viruses, imported this system (for example WO2006/084746) in retroviral.In addition, about the paramyxovirus coe virus under human parainfluenza 2 C-type virus Cs, the sendai virus vector (WO2000/070070) of the defects such as F gene has been proposed.In addition, report, find that following tuberculosis morbidity vaccine has strong tubercule bacillus proliferation inhibiting effect, described tuberculosis morbidity vaccine is with the non-proliferative gene recombination type carrier (PCT/JP2010/069435) of human parainfluenza virus's 2 C-type virus Cs of α antigen (Ag85B) gene of having introduced the mycobacterium kansasii (Mycobacterium kansasii) that carrys out the self-fixing acid fast bacteria or bacillus tuberculosis bovis (Mycobacterium bovis) BCG.
There is not the fusion rotein (below be designated as " F albumen ") of human parainfluenza 2 C-type virus Cs in F gene defection type human parainfluenza 2 C-type virus Cs, this fusion rotein be separately by invade this virus after host cell copy transcription step, make peplos and cytolemma merge, to necessary during importing virus nucleocapsid albumen in the host, therefore, F gene defection type human parainfluenza 2 C-type virus Cs can not form the virion that keeps the autonomous proliferation ability.Therefore, although with regard to F genetic flaw on the preparation genome but contain on the carrier coating with regard to the non-proliferous type human parainfluenza 2 C-type virus C carriers of F albumen, express the cell of this virus F protein, cultivate this defective virus in this cell by manufacturing, in the situation that from the F albumen of the trans supply of this cell, exist, make F albumen remain on peplos, thereby build the virion with infection ability.The genome of the human parainfluenza 2 C-type virus C particles that reclaim from the culture supernatant by the preparation of this proliferating system, the F gene lacks., when in advance when introducing treatment with the gene of gene, encoding vaccine antigens in these F gene defection type human parainfluenza 2 C-type virus C carriers, can obtain as the useful virion of pharmaceuticals.
Generally the membrane protein of virus has cytotoxicity, therefore must set up cell strain by import the carrier that the mode expressed with virus F gene etc. designs under inducible promoter is controlled in the past, usually suppress the expression of F gene etc. the time, only at virus infection, express the helper of F gene etc. and just make while being reconstructed F gene etc. be induced to express.For example, in the prior art document (WO2000/070070) relevant to above-mentioned sendai virus vector, used following system: wherein use the Cre-loxP inducible system, during host cell propagation, the F gene of Sendai virus is not expressed, and by add adenovirus when cell infection is viral, carrys out abduction delivering.
But, in the business of virus vector is made, prepares must be arranged and adds the step of adenovirus, therefore operate miscellaneously, in addition, the gene importing efficiency by adenovirus neither be certain, so have the problem of pharmaceuticals manufacturing management step complexity.And, in the induced expression system of reality use membrane protein is made the step of virus vector, culturing cell makes in the situation of virion propagation, there is following problems: because express membrane protein, have toxicity, along with host cell is gone down to posterity, the multiplication capacity of virus reduces, and through about going down to posterity of 5 generations, the generation ability of virus has just disappeared substantially.
Therefore, as the gene that can express the membrane protein etc. of defect in coding virus and make the cell of defective virus carrier propagation, people are seeking stably to express consistently the clone of this albumen.And people at the character host cell constant, that have fixing afterwards of seeking to go down to posterity are.
The reference of quoting in this specification sheets as shown below.The content of putting down in writing in these documents is all by reference to incorporating this specification sheets into.These documents all are not considered to the prior art with respect to this specification sheets.
Patent documentation 1:WO94/28152
Patent documentation 2:WO2006/084746
Patent documentation 3:WO2000/070070
Patent documentation 4:PCT/JP2010/069435
Summary of the invention
The object of the invention is to the cell as the virus multiplication of the F genetic flaw that makes human parainfluenza 2 C-type virus Cs, provide a kind of and can stably express consistently the clone of the F albumen of human parainfluenza 2 C-type virus Cs, and the method for utilizing above-mentioned cell to make F gene defection type human parainfluenza 2 C-type virus C carriers is provided.
The discovery Vero cells such as the present inventor, as the packing cell that can infect human parainfluenza virus's 2 C-type virus C F genetic flaw C-type virus Cs, generation virion, have outstanding character.Unexpectedly confirm, the Vero cell is high especially to the admissibility of human parainfluenza 2 C-type virus C F albumen, do not express in addition Interferon, rabbit, even in the situation that make the constant expression of F gene of human parainfluenza 2 C-type virus Cs can stablize propagation, and can efficiently produce virion yet.
That is, the invention provides the manufacture method of non-proliferous type human parainfluenza 2 C-type virus C carriers.The method comprises following each step:
With the human parainfluenza of F genetic flaw 2 C-type virus Cs and Vero co-culture of cells, described Vero cell has the form of the F gene of non-inducible expression's human parainfluenza 2 C-type virus Cs, then separating viral particles from culture supernatant.
Method of the present invention preferred embodiment in, to introducing vaccine in the gene of human parainfluenza 2 C-type virus Cs of F genetic flaw with gene or treatment gene.
, according to other viewpoints, the invention provides the Vero cell that has the F gene of human parainfluenza 2 C-type virus Cs with non-inducible expression's form.
The method of the application of the invention, can stablize, make efficiently the human parainfluenza 2 C-type virus C carriers of non-proliferous type.
Description of drawings
[Fig. 1] Fig. 1 represents constitution and the genome of human parainfluenza 2 C-type virus Cs.
[Fig. 2] Fig. 2 represents: confirm F genetic expression in the F genetic expression Vero cell of after long-term cultivation (about 1 year) by the RT-PCR method, the expression of F gene is maintained.
[Fig. 3] Fig. 3 represents Western trace figure, wherein confirms at the F gene and imports F protein expression in the Vero cell.
[Fig. 4] Fig. 4 represents to contain the part of the plasmid structure of the inverted defined gene group cDNA of human parainfluenza 2 C-type virus Cs (disappearance F gene) and GFP gene.
[Fig. 5] Fig. 5 represents Western trace figure, wherein confirms the hPIV2 that maintains M2 gene, F genetic flaw and expresses M2 albumen.
Embodiment
The invention provides and use Vero cell (the F gene that has human parainfluenza 2 C-type virus Cs with non-inducible expression's form), make the method for the human parainfluenza 2 C-type virus C propagation of F genetic flaw.
Human parainfluenza 2 C-type virus Cs are the virus that belongs to Paramyxoviridae, and its genome is the negative RNA of the monocistron strand of approximately 15,000 bases.Fig. 1 shows the basic granules structure of human parainfluenza 2 C-type virus Cs (hPIV2).Nucleocapsid protein (NP) is bonded to this nucleic acid, forms helical symmetry ribonucleoside protein complexes (nucleocapsid RNP).In the protein of virogene group coding, (large, Large) albumen is that the formation of RNP is necessary for NP albumen, P (phosphoric acid) albumen and L.F (fusion) albumen and HN (hemagglutinin neuraminidase) albumen are envelope proteins, and it is important for the infection to cell.M (matrix) albumen is the membranin of support package membrane structure.
Human parainfluenza 2 C-type virus Cs are the RNA viruses of breeding in the tenuigenin of cells infected, so gene is not introduced in the karyomit(e) of host cell.In addition, thereby known this virus infection people tracheae mucous membrane, by inducing the expression mucosa immunity-inducing of IgA, and the severe report case that there is no up to now the adult, so consider to be extremely useful as vaccine with virus vector or therapeutic viruses carrier with it.
Human parainfluenza 2 C-type virus Cs are changed into and expressed NP albumen, P albumen and L albumen but while not expressing F albumen, virion energy cells infected 1 time, but can not produce the virion with infection ability in this cell.Therefore, have advantages of safe with virus vector as the treatment vaccine.
The F protein expression Vero cell of human parainfluenza 2 C-type virus Cs of the present invention, be useful host cell, and it can be used for making so virus genomic F gene defection type virus multiplication, makes the virion of F genetic flaw.
" Vero cell " is widely used cell strain in the whole world, and it is the culturing cell that is derived from the kidney epithelium of cercopithecus aethiops, has the feature that does not produce Interferon, rabbit, and be multiplex in the virus infection experimental vaccine is produced etc.In the present invention, also can use any commercially available Vero cell.
" F gene " is the gene of the F albumen of encoding human parainfluenza 2 C-type virus Cs, and this term in this specification sheets is not limited to the geneome RNA of virogene, also is used for expression and has corresponding sequence or any DNA and the RNA of its complementary sequence.
" non-inducible expression " refers to be designed to not is the such expression of inducible expression, do not apply induce the operation and express.Herein, inducible expression represents the following system that combines by host/vector and culture condition, does not express when it is designed to not carry out inducing operation or while suppressing operation and only when having carried out inducing operation or removed expression while suppressing operation.Multiple inducible expression known in the art.Non-inducible expression represents not carry out inducing operation or does not suppress the releasing that operates and express.Typically, although non-inducible expression is constant expression, but the expression of " non-inducibility " in the present invention is also contained in the specific period of cell fission, cell cycle and expresses repressed situation, and the culture condition such as culture temperature, substratum compositions, cell density cause the repressed situation of expression.
In order to make F protein expression Vero cell of the present invention, at first preparation has the recombinant plasmid vector of F gene and mark (for example medicament patience), and the Vero cell is advanced in its transfection.The sequence of F gene is known,, by the F gene is introduced commercially available suitable plasmid vector, can easily make recombinant plasmid vector.With regard to transfection, can use various commercially available transfections with reagent or by electroporation, carry out according to existing method.Then, utilize mark to identify transformant, separate and enlarged culturing.Can analyze the expression of F albumen in the Vero cell that transforms by following method: carry out the expression of analysing protein level by the immunostaining of antibody, as shown in hereinafter embodiment by the Western trace or analyze the expression of rna level by RT-PCR etc.Perhaps, also can form by the polykaryon in the cytogamy of observing cells infected, confirm the expression of F albumen." polykaryon formation " refers to: the co expression in same cell by F albumen and receptor binding protein HN (it is the different membrane protein of virus), the giant cell that the cytogamy of adjacency, a plurality of nucleus of formation are assembled.The plasmid transfection that will contain the HN gene advances expection and can import the Vero cell of F gene, if can confirm that polykaryon forms, and just can confirm this cell function expression F gene.Thus, can the desired restructuring Vero that express the F gene be cloned.
With regard to other viewpoints of the present invention, provide the manufacture method of the non-proliferous type human parainfluenza 2 C-type virus C carriers of F genetic flaw.The method contains following each step: the human parainfluenza of F genetic flaw 2 C-type virus Cs and form with non-inducible expression of the present invention are had the Vero co-culture of cells of the F gene of human parainfluenza 2 C-type virus Cs, then from the culture supernatant separating viral particles.
" virus vector " refers to: at the gene that should express in the cell that has infected packaged virion that forms together with the viral genome gene.
The viral genome gene of F genetic flaw can followingly build: use habitual gene recombination technology, from containing the plasmid of the Antisense cDNA corresponding with the full genomic gene of hPIV2, make all or part of zone disappearance of F gene or import the terminator codon sudden change in the part of F gene, building thus.Cause obtaining once again the F gene function for fear of the sudden change suddenly of the virus vector owing to giving the experimenter, preferably the F gene is all lacked.More preferably, non-proliferous type virus vector of the present invention is designed to contain be useful on and introduces the cloning site of various treatments with gene.
For the viral genome gene by the F genetic flaw is made F gene defection type virion, to build the plasmid that forms in the viral genome gene of F genetic flaw is expressed under the control of T7 promotor mode, transfection advances to express the cell of t7 rna polymerase together with 4 kinds of carriers of the F albumen of expressing hPIV2 and polysaccharase unit (NP albumen, P albumen and L albumen), or together with the plasmid of expressing the T7 polysaccharase, the polysaccharase unit of hPIV2 (NP albumen, P albumen and L albumen) transfection is advanced to express the Vero of F gene.Cultivate the cell of infection after 3～6 days, can reclaim F gene defection type virion from culture supernatant.
While with the F gene defection type virion that obtains thus, making the Vero cell infection of expression F gene of the present invention, virion is in the cell internal breeding,, although be F gene defection type virion, himself be created on peplos the infectious viral particle with F albumen.
In a kind of preferred implementation of the present invention, introduce in the virus vector of human parainfluenza 2 C-type virus Cs based on the F genetic flaw and treat with the vaccine gene,, with the non-proliferous type viral vector infection target cell that the method according to this invention obtains, can carry out thus to import the treatment gene in the target cell.Treatment is the gene that should express in the cell that infects with gene, as an example, can enumerate coding from the gene of the gene of the mammiferous albumen that comprises the people or its part, coding cancer antigen or its part, from the gene of bacterium or virus, coding treatment with the gene of antibody or its part, and the fragment of these genes and to the gene that has imported sudden change in these genes.Contain from the gene of bacterium or virus or the virus vector of its fragment and can be used as the vaccine use., for introducing the treatment gene in the gene to human parainfluenza 2 C-type virus Cs, can learn a skill to carry out according to existing method, habitual recombinant DNA technology and the reverse genetics of use.
Typically, virus vector constructed in accordance can be used as sprays and comprises people's mammalian cell.Sprays can prepare by existing method.For example, can be prepared by following method: as required the culture supernatant that contains virus vector is concentrated, it is suspended in the damping fluids such as PBS or physiological saline together with suitable carrier or vehicle,, as required with filtration sterilizations such as strainers, then be filled into sterile chamber afterwards.Can also add stablizer, preservatives etc. as required in sprays.The expression vector that obtains thus can carry out inhalation to the experimenter.
While using in this manual, the form of expressing by " comprising/contain/comprise (comprising) " such statement comprise with " basically by ... form (essentially consisting of) " the represented form of statement and with " by ... form (consisting of) " the represented form of statement.
In this specification sheets, the content of all patents of specific reference and reference is all by reference to introducing in this specification sheets.
Embodiment
Hereinafter by embodiment, illustrate in greater detail the present invention, but the present invention is not subjected to the restriction of these embodiment.
The manufacturing of embodiment 1F genetic expression Vero cell
To maintain Liu Suanyan NEOMYCIN SULPHATE tolerance gene (Neo) and maintain bird β actin promoter sequence and the plasmid of rabbit beta Globulin polyA sequence in import the F gene cut out from the genomic gene of hPIV2, build plasmid pCXN2-F.In contrast, use plasmid pCAL-F, it is built as and can expresses the F gene by the Cre-loxP inducible system.Use the Nucleofector of amaxa company, above-mentioned plasmid transfection is advanced the Vero cell.
Cultivate the cell through transfection in the substratum that contains Liu Suanyan NEOMYCIN SULPHATE (1mg/m1), by Liu Suanyan NEOMYCIN SULPHATE medicament patience, screen.Go out 20 strain medicament patience bacterium colonies from the cellular segregation that has imported pCXN2-F, from the cellular segregation that has imported pCAL-F, go out 27 strain medicament patience bacterium colonies, analyze the expression of F gene.At this moment, in the situation of pCAL-F as plasmid, infect the adenovirus that keeps the Cre gene,, by with the loxP sequence, removing unwanted gene segment, induce F genetic expression.
Then, confirmed polykaryon formation.The plasmid SRa-HN that make to keep the HN gene, use Nucleofector, in the Liu Suanyan NEOMYCIN SULPHATE patience Vero cell that this plasmid transfection is advanced to obtain.Examine under a microscope polykaryon after 1 day or 2 days and form, to forming coenocytic cell, as F genetic expression cell, carry out postsearch screening.In isolated 20 clones, l2 clone shown the ability that forms multinuclear the cell from having imported pCXN2-F.
Then, measure the expression of F gene by RT-PCR on rna level.As its result, in isolated 20 clones, 12 clones have shown the ability that forms multinuclear from the cell that has imported pCXN2-F, and wherein 9 are cloned in RT-PCR positive.In addition, in isolated 27 clones, 9 clones have shown the ability that forms multinuclear from the cell that has imported pCAL-F, and wherein 7 are cloned in RT-PCR positive.In addition, use by pCXN2-F import the F gene and cultivated 1 year 2 * 10
6Individual Vero cell carries out single step (One-Step) RT-PCR (QIAGEN).Exist in the virus genomic situation of F gene defection type, primer is set as near acquisition pcr amplification product 550bp carries out PCR., as its result, in the PCR that carries out respectively for 3 different clones, confirm the band of pcr amplification product near 550bp.Can confirm that the F gene is still in expression (Fig. 2).That is, efficiency that can be suitable with the Vero cell with abduction delivering F gene obtains the Vero cell of constant expression F gene, and continuous expression long-term and stably.This shows: the Vero cell is high to the admissibility of F albumen, even express for a long time consistently in the situation of F gene, also can stably breed.
Then, the F protein expression in F genetic expression Vero cell is confirmed.For 9.5 * 10
5Individual F genetic expression Vero cell, the 0.03%Sulfo-NHS-LC-biotin solution of use 1ml, all albumen that exist on cell membrane carry out biotinylation, make the cell pyrolysis liquid of 500 μ l under mild conditions.Then the F protein-specific antibody with 80 μ l carries out immunosedimentation to 300 μ l cell pyrolysis liquids, and only F albumen and antibodies, carry out selective recovery, concentrated to it.This sample is all carried out SDS-PAGE, be transferred on pvdf membrane.At this moment, F albumen has become by biotinylated state, by Avidin-Biotin complex body (avidin-biotin complex) method, forms the Avidin-Biotin complex body.Because avidin is by horseradish peroxidase (HRP) mark mistake, so with ECL reagent react, luminous detection by HRP, arrived by biotinylated F albumen (Fig. 3).
The genomic structure of embodiment 2F gene defection type antisense hPIV2
Make the sequence that contains whole F genes encodings zone from disappearance fully on plasmid (this plasmid contains Antisense cDNA corresponding to the full genomic gene of hPIV2 in T7 promotor downstream), built introduced GFP (green fluorescent protein) gene at the NotI restriction site plasmid (hPIV2-Δ F-GFP) (Fig. 4).Build following carrying out: use the plasmid of the genomic gene that contains hPIV2 as template, use based on the primer of the sequences Design of F upstream area of gene and downstream area and the GFP gene segment that cuts out from the commercially available carrier that contains GFP, by multistage PCR, undertaken.
Use Lipofectamine or FUGENE6 as transfection reagent, plasmid (the hPIV2-Δ F-GFP) transfection of making in embodiment 2 is advanced to express the cell of t7 rna polymerase.At this moment, together transfection express the 4 kinds of carriers that amount to of the F albumen of hPIV2 and polysaccharase unit (NP albumen, P albumen and L albumen).
Cultivate after 3 days, reclaim supernatant liquor, infect F genetic expression Vero cell or do not express the common Vero cell of F gene, infects after 6 days observation GFP fluorescence.For F genetic expression Vero cell, cell all confirms GFP fluorescence.And on the other hand, for common Vero cell, the cell quantity that presents GFP fluorescence is compared and is wanted much less with F genetic expression Vero cell.
Then, reclaim the culture supernatant of the F genetic expression Vero cell that presents GFP fluorescence, infect other F genetic expression Vero cell on culture dish, reclaim this culture supernatant.After repeating this operation 3 times, get the 1.5ml culture supernatant, with 1000x g centrifugal 1 minute, remove residue.With supernatant liquor with 20,000x g centrifugal 30 minutes, remove supernatant liquor, throw out is suspended in 20 μ l does not contain in the water of RNAase.Get 0.5 μ l, it is carried out single step RT-PCR (QIAGEN).The result of PCR,, with in the situation that exist in the virus genomic situation of F gene defection type and have approximately 400bp, at the GFP gene, insert and have the approximately mode of 500bp, set respectively primer.As its result, in minute other PCR, in confirming the band of pcr amplification product near 400bp and near 500bp.Think that thus the PIV2 of recyclable F gene defection type is viral.
The GFP that obtains is as mentioned above expressed the strainer of the culture supernatant of F genetic expression Vero cell by 0.45 μ m footpath, removed residue.This culture supernatant is sequentially carried out 10 times of dilutions, infect the Vero cell of not expressing the F gene.Confirmed the cell of the fluorescence that presents GFP after 3 days.As its result, found to present the cell of GFP fluorescence, the quantity of GFP positive cell reduces pro rata with dilution.Confirm thus in the culture supernatant of F genetic expression Vero cell and have F gene defection type hPIV2 virus, this virus has infected the Vero cell of not expressing the F gene.It should be noted that in the culture supernatant of the Vero cell of never expressing the F gene and do not obtain to have infective virion.
In addition, after the hPIV2 virus infected mice NIH-3T3 cell with the F genetic flaw that keeps the GFP gene, although observe the existence of the positive NIH-3T3 cell of GFP, efficiency of infection is lower than Vero cell.
The F of the viral M2 albumen of embodiment 4 expression of influenza viruses (influenza virus)
The manufacturing of gene defection type PIV2 virus
With embodiment 2 in the same manner, be structured in the genomic NotI restriction site of F gene defection type antisense hPIV2 and introduced the plasmid (hPIV2-Δ F-M2) of M2 albumen (M2 ionic channel) gene on the viral lipid film that is present in influenza virus.With embodiment 3 in the same manner, use Lipofectamine or FUGENE6 as transfection reagent, transfection advances to express the cell of t7 rna polymerase.At this moment, together transfection express the 4 kinds of carriers that amount to of the F albumen of hPIV2 and polysaccharase unit (NP albumen, P albumen and L albumen).Then, carry out virus with the method identical with embodiment 3 and reclaim, infect the Vero cell (2 * 10 of having expressed the F gene with the hPIV2 that keeps M2 gene and F genetic flaw
6Individual), reclaim cell after 2 days, use M2 antibody to carry out the Western engram analysis to it., as its result, can confirm the expression (Fig. 5) of M2.That is,, even confirm gene except GFP, also can reclaim the PIV2 virus of F gene defection type.
From the above results as can be known, express the Vero cell of F gene by use, the non-proliferous type hPIV2 virus of recyclable F genetic flaw, this virus can only be bred in F genetic expression cell, although can infect in the cell of not expressing the F gene 1 time, not produce infectious virus.
Utilize possibility on industry
The present invention can be used for the manufacturing of the virus vector that gene therapy uses.
Claims (3)
1. method of making non-proliferous type human parainfluenza 2 C-type virus C carriers, described method comprises following each step:
With the human parainfluenza of F genetic flaw 2 C-type virus Cs and Vero co-culture of cells, described Vero cell has the F gene of human parainfluenza 2 C-type virus Cs with non-inducible expression's form,
Then separating viral particles from culture supernatant.
2. the method for claim 1, wherein introduced vaccine with gene or treatment gene in the gene of human parainfluenza 2 C-type virus Cs of F genetic flaw.
3.Vero cell, its form with non-inducible expression has the F gene of human parainfluenza 2 C-type virus Cs.
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| PCT/JP2012/000838 WO2012108195A1 (en) | 2011-02-08 | 2012-02-08 | Method for producing virus vector for gene transfer |
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| EP (1) | EP2674491B1 (en) |
| JP (1) | JP5807917B2 (en) |
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| US20180339038A1 (en) * | 2015-06-12 | 2018-11-29 | Mie University | Human parainfluenza virus type 2 vector and vaccine |
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| CN1856575A (en) * | 2000-04-28 | 2006-11-01 | 圣朱德儿童研究医院 | DNA transfection system for the generation of infectious influenza virus |
| CN1871355A (en) * | 2003-06-09 | 2006-11-29 | 惠氏 | Method for the recovery of non-segmented, nagative-stranded rna viruses from cdna |
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| CA2499404A1 (en) | 2002-09-18 | 2004-04-01 | The United States Of America As Represented By The Secretary Of The Department Of Health & Human Services | Recovery of recombinant human parainfluenza virus type 2 (hpiv2) from cdna and use of recombinant hpiv2 in immunogenic compositions and as vectors to elicit immune responses against piv and other human pathogens |
| JP2008537683A (en) * | 2005-01-12 | 2008-09-25 | ザ ガバメント オブ ザ ユナイテッド ステイツ オブ アメリカ，レプリゼンテッド バイ ザ セクレタリー，デパートメント オブ ヘルス アンド ヒューマン サービシズ | Attenuated human parainfluenza virus, methods and uses thereof |
| DE102005006388A1 (en) | 2005-02-11 | 2006-08-24 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Replication-deficient RNA viruses as vaccines |
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| WO2011052771A1 (en) | 2009-11-02 | 2011-05-05 | 国立大学法人三重大学 | Nebulizable tuberculosis vaccine for transnasal administration comprising paramyxovirus vector |
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| CN1856575A (en) * | 2000-04-28 | 2006-11-01 | 圣朱德儿童研究医院 | DNA transfection system for the generation of infectious influenza virus |
| CN1646684A (en) * | 2002-02-21 | 2005-07-27 | 免疫医疗疫苗公司 | Recombinant parainfluenza virus expression systems and vaccines comprising heterologous antigens derived from metapneumovirus |
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| EP2674491A4 (en) | 2016-05-25 |
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| CN103403159B (en) | 2018-09-14 |
| ES2640113T3 (en) | 2017-10-31 |
| EP2674491B1 (en) | 2017-06-14 |
| EP2674491A1 (en) | 2013-12-18 |
| US20140322760A1 (en) | 2014-10-30 |
| JP5807917B2 (en) | 2015-11-10 |
| WO2012108195A1 (en) | 2012-08-16 |
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